U.S. patent application number 12/640509 was filed with the patent office on 2010-04-15 for method of measuring microparticles having nucleic acid and apparatus therefor.
This patent application is currently assigned to Toyohashi University of Technology. Invention is credited to Akira MIZUNO, Masudur Rahman, Kazunori Takashima, Hachiro Yasuda.
Application Number | 20100089754 12/640509 |
Document ID | / |
Family ID | 40156293 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100089754 |
Kind Code |
A1 |
MIZUNO; Akira ; et
al. |
April 15, 2010 |
METHOD OF MEASURING MICROPARTICLES HAVING NUCLEIC ACID AND
APPARATUS THEREFOR
Abstract
To provide a system by which evaluation of circumstances of
contamination by microparticles having nucleic acid can be
performed rapidly and accurately. The theme is achieved by a system
for measuring microparticles that includes: (1) a microparticle
adhesion step of adhering the microparticles having nucleic acid to
a microparticle adhesion member; (2) a membrane breakage step of
breaking membranes of the adhered microparticles by electrical
discharge; (3) an electrophoresis step of electrophoresing the
microparticles in a thickness direction of a gel to make the
nucleic acid in the microparticles migrate from a negative
electrode side toward a positive electrode side and adhere the
nucleic acid on a surface of a nucleic acid detection member; and
(4) a nucleic acid measurement step of fluorescently staining the
surface of the nucleic acid detection member to measure a
concentration of the nucleic acid.
Inventors: |
MIZUNO; Akira;
(Toyohashi-Shi, JP) ; Takashima; Kazunori;
(Toyohashi-Shi, JP) ; Yasuda; Hachiro;
(Toyohashi-Shi, JP) ; Rahman; Masudur;
(Toyohashi-Shi, JP) |
Correspondence
Address: |
BURR & BROWN
PO BOX 7068
SYRACUSE
NY
13261-7068
US
|
Assignee: |
Toyohashi University of
Technology
Toyohashi-Shi
JP
|
Family ID: |
40156293 |
Appl. No.: |
12/640509 |
Filed: |
December 17, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2008/061210 |
Jun 19, 2008 |
|
|
|
12640509 |
|
|
|
|
Current U.S.
Class: |
204/461 ;
204/613; 204/614 |
Current CPC
Class: |
C12Q 1/6825 20130101;
G01N 27/44743 20130101; C12Q 1/6825 20130101; C12Q 2523/307
20130101; C12Q 2565/125 20130101; C12Q 2523/307 20130101; C12Q
2563/107 20130101; C12Q 2565/125 20130101; C12Q 2563/107 20130101;
C12Q 1/6834 20130101; C12Q 1/6834 20130101 |
Class at
Publication: |
204/461 ;
204/613; 204/614 |
International
Class: |
G01N 33/559 20060101
G01N033/559; C07K 1/26 20060101 C07K001/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2007 |
JP |
2007-162261 |
Claims
1. A method of measuring microparticles having nucleic acid
comprising: (1) a microparticle adhesion step of adhering the
microparticles having nucleic acid to a microparticle adhesion
member; (2) a membrane breakage step of breaking membranes of the
adhered microparticles by electrical discharge; (3) an
electrophoresis step of electrophoresing the microparticles in a
thickness direction of a gel to make the nucleic acid in the
microparticles migrate from a negative electrode side toward a
positive electrode side and adhere the nucleic acid on a surface of
a nucleic acid detection member; and (4) a nucleic acid measurement
step of fluorescently staining the surface of the nucleic acid
detection member to measure a concentration of the nucleic
acid.
2. The method of measuring microparticles having nucleic acid
according to claim 1, wherein in the microparticle adhesion step,
the microparticle adhesion member is placed on a surface at a
smooth electrode side of a corona discharge electrode, and
microparticles having nucleic acid that are suspended in air are
adhered to the surface of the microparticle adhesion member.
3. The method of measuring microparticles having nucleic acid
according to claim 1, wherein in the membrane breakage step, an
electrode system, in which an insulating plate is inserted between
mutually opposing electrodes, is used, the microparticle adhesion
member is inserted in a gap between the electrodes, and the
membranes of the microparticles are broken by applying an
alternating voltage across the electrodes.
4. The method of measuring microparticles having nucleic acid
according to claim 1, wherein the surface of the nucleic acid
detection member to which the nucleic acid is electrophoresed has a
positive surface charge.
5. A system for measuring microparticles having nucleic acid
comprising: a microparticle adhesion member on which the
microparticles having nucleic acid are adhered; a microparticle
adhesion apparatus making the microparticles having nucleic acid
adhere to a surface of the microparticle adhesion member; a
membrane breakage apparatus using electrical discharge to break
membranes of the microparticles that are adhered to the
microparticle adhesion member; a nucleic acid detection member in
turn comprising a tape, including a charge neutralizer, and a gel,
disposed on one surface side of the tape; an electrophoresis
apparatus electrophoresing the nucleic acid, adhered to the surface
of the microparticle adhesion member, through the gel of the
nucleic acid detection member to make the nucleic acid migrate to
the tape side; and a nucleic acid detection apparatus detecting the
nucleic acid on the tape surface in a state where the gel of the
nucleic acid detection member is removed.
6. The system for measuring microparticles having nucleic acid
according to claim 5, wherein the microparticle adhesion apparatus
comprises: a corona discharge electrode having a needle electrode
and a flat plate electrode; and makes the microparticles adhere to
the surface with the microparticle adhesion member being set on the
flat plate electrode.
7. The system for measuring microparticles having nucleic acid
according to claim 5, wherein the membrane breakage apparatus
comprises: mutually opposing electrodes; an insulating plate
inserted between the electrodes; and a member mounting space into
which the microparticle adhesion member is inserted; and breaks the
membranes by applying an alternating voltage across the
electrodes.
8. The system for measuring microparticles having nucleic acid
according to claim 5, wherein the tape of the nucleic acid
detection member has a positive surface charge.
9. The system for measuring microparticles having nucleic acid
according to claim 5, wherein the nucleic acid detection member
serves in common as the microparticle adhesion member, and a
microparticle membrane breaking operation is performed in the state
where the microparticles having nucleic acid are adhered to the
surface of the gel.
10. The method of measuring microparticles having nucleic acid
according to claim 2, wherein in the membrane breakage step, an
electrode system, in which an insulating plate is inserted between
mutually opposing electrodes, is used, the microparticle adhesion
member is inserted in a gap between the electrodes, and the
membranes of the microparticles are broken by applying an
alternating voltage across the electrodes.
11. The method of measuring microparticles having nucleic acid
according to claim 2, wherein the surface of the nucleic acid
detection member to which the nucleic acid is electrophoresed has a
positive surface charge.
12. The method of measuring microparticles having nucleic acid
according to claim 3, wherein the surface of the nucleic acid
detection member to which the nucleic acid is electrophoresed has a
positive surface charge.
13. The system for measuring microparticles having nucleic acid
according to claim 6, wherein the membrane breakage apparatus
comprises: mutually opposing electrodes; an insulating plate
inserted between the electrodes; and a member mounting space into
which the microparticle adhesion member is inserted; and breaks the
membranes by applying an alternating voltage across the
electrodes.
14. The system for measuring microparticles having nucleic acid
according to claim 6, wherein the tape of the nucleic acid
detection member has a positive surface charge.
15. The system for measuring microparticles having nucleic acid
according to claim 7, wherein the tape of the nucleic acid
detection member has a positive surface charge.
16. The system for measuring microparticles having nucleic acid
according to claim 6, wherein the nucleic acid detection member
serves in common as the microparticle adhesion member, and a
microparticle membrane breaking operation is performed in the state
where the microparticles having nucleic acid are adhered to the
surface of the gel.
17. The system for measuring microparticles having nucleic acid
according to claim 7, wherein the nucleic acid detection member
serves in common as the microparticle adhesion member, and a
microparticle membrane breaking operation is performed in the state
where the microparticles having nucleic acid are adhered to the
surface of the gel.
18. The system for measuring microparticles having nucleic acid
according to claim 8, wherein the nucleic acid detection member
serves in common as the microparticle adhesion member, and a
microparticle membrane breaking operation is performed in the state
where the microparticles having nucleic acid are adhered to the
surface of the gel.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method and an apparatus
related to measurement of microparticles having nucleic acid for
ascertaining circumstances of contamination by microorganisms,
etc., of air, wall surfaces, and floors of hospitals and food
factories or utensils and clothing used for surgery and food
manufacturing.
BACKGROUND OF THE INVENTION
[0002] From before, there have been needs to examine circumstances
of contamination by microorganisms in air and on wall surfaces and
floor surfaces of hospitals, food factories, and other locations
where particular attention must be paid to sanitation as well as to
examine circumstances of contamination by microorganisms of
utensils and clothing used for surgery and food manufacturing
(Non-Patent Document 1). In a conventional method, a sample is
coated as a solution onto a culture medium or a membrane having a
plurality of cells fixed thereon, formation of colonies by a
microorganism or loss of the cells spread in the form of the
membrane due to a virus is made to occur, and evaluation is
performed by counting the number of colonies or number of loss
(Non-Patent Document 2).
[0003] Meanwhile, by advances made in single-molecule DNA
manipulation and measurement arts through progress of technical
development in recent years, it has become possible to make clear
observations by fluorescently staining of single-molecule DNA
(Non-Patent Document 3). Using this art, it is becoming possible to
take out a DNA molecule from a cell, extend and fix the DNA
molecule on a substrate, and adhere a fluorescently stained
restriction enzyme to the extended and fixed DNA to prepare a
restriction map using a fluorescence microscope (Patent Document 1
and Non-Patent Document 4). [0004] Patent Document 1: Japanese
Published Unexamined Patent Application No. 2003-200400 [0005]
Non-Patent Document 1: U Yanagi: "Behavior of bio-aerosol in office
buildings and methods of control," Green Technology, vol. 17, No.
5, 44-47, 2007 [0006] Non-Patent Document 2: Sumiyo Ishimatsu:
"Capture and detection of airborne microorganism particles," Green
Technology, vol. 17, No. 5, 48-51, 2007 [0007] Non-Patent Document
3: Morikawa K., and Yanagida M., J. Biochem., 89, pp. 693-696, 1981
[0008] Non-Patent Document: A. Bensimon, A. Simon, A. Chiffaudel,
V. Croquette, F. Heslot, D. Bensimon, "Alignment and sensitive
detection of DNA by a moving interface," Science, Vol. 265(5181),
pp. 2096-2098, 1994
SUMMARY OF THE INVENTION
[0009] With the present invention, a method of visualizing a single
molecule is applied to detection of particles having nucleic acid
to rapidly and accurately count microorganisms in an
environment.
[0010] Conventionally, evaluation of circumstances of contamination
by microorganisms in an environment is carried out by colony
formation or cell loss by culturing. However, a time of several
days is required to culture cells or to infect cells with a virus
to cause cell loss. Even with a method of using a fluorescent dye
to stain and observe DNA in a cell, a cell membrane is not high in
dye permeability and thus a staining process time of not less than
approximately ten hours is required (Non-Patent Document 2).
Frequently in this process, organelles inside the cell are
fluorescently stained at the same time, and this has been a cause
of error. When fluorescent staining is performed upon breaking the
cell membrane to reduce the processing time, biopolymers besides
the nucleic acid become stained and this becomes a cause of
error.
[0011] As a result of repeating diligent research in view of the
above circumstances, the present inventor has come to complete the
following invention.
[0012] A method of measuring microparticles having nucleic acid
according to a first aspect of the present invention includes: (1)
a microparticle adhesion step of adhering the microparticles having
nucleic acid to a microparticle adhesion member; (2) a membrane
breakage step of breaking membranes of the adhered microparticles
by electrical discharge; (3) an electrophoresis step of
electrophoresing the microparticles in a thickness direction of a
gel to make the nucleic acid in the microparticles migrate from a
negative electrode side toward a positive electrode side and adhere
the nucleic acid on a surface of a nucleic acid detection member;
and (4) a nucleic acid measurement step of fluorescently staining
the surface of the nucleic acid detection member to measure a
concentration of the nucleic acid.
[0013] Preferably, in the present invention, in the microparticle
adhesion step, the microparticle adhesion member is placed on a
surface at a smooth electrode side of a corona discharge electrode
and microparticles having nucleic acid that are suspended in air
are adhered to the surface of the microparticle adhesion
member.
[0014] Also, preferably in the membrane breakage step, an electrode
system, in which an insulating plate is inserted between mutually
opposing electrodes, is used, the microparticle adhesion member is
inserted in a gap between the electrodes, and the membranes of the
microparticles are broken by applying an alternating voltage across
the electrodes.
[0015] Also, preferably, the surface of the nucleic acid detection
member to which the nucleic acid is electrophoresed has a positive
surface charge.
[0016] A system for measuring microparticles having nucleic acid
according to a second aspect of the present invention includes: a
microparticle adhesion member on which the microparticles having
nucleic acid are adhered; a microparticle adhesion apparatus making
the microparticles having nucleic acid adhere to a surface of the
microparticle adhesion member; a membrane breakage apparatus using
electrical discharge to break membranes of the microparticles that
are adhered to the microparticle adhesion member; a nucleic acid
detection member in turn including a tape, including a charge
neutralizer, and a gel, disposed on one surface side of the tape;
an electrophoresis apparatus electrophoresing the nucleic acid,
adhered to the surface of the microparticle adhesion member,
through the gel of the nucleic acid detection member to make the
nucleic acid migrate to the tape side; and a nucleic acid detection
apparatus detecting the nucleic acid on the tape surface in a state
where the gel of the nucleic acid detection member is removed.
[0017] Preferably, in the present invention, the microparticle
adhesion apparatus includes: a corona discharge electrode having a
needle electrode and a flat plate electrode; and makes the
microparticles adhere to the surface with the microparticle
adhesion member being set on the flat plate electrode.
[0018] Also preferably, the membrane breakage apparatus includes:
mutually opposing electrodes; an insulating plate inserted between
the electrodes; and a member mounting space into which the
microparticle adhesion member is inserted; and breaks the membranes
by applying an alternating voltage across the electrodes.
[0019] Also preferably, the tape of the nucleic acid detection
member has a positive surface charge.
[0020] Also preferably, the nucleic acid detection member serves in
common as the microparticle adhesion member, and a microparticle
membrane breaking operation is performed in the state where the
microparticles having nucleic acid are adhered to the surface of
the gel.
[0021] The nucleic acid may be either RNA or DNA. The nucleic acid
may be either single-stranded or double-strand and may be cyclic or
linear.
[0022] The microparticles having nucleic acid refer to those having
nucleic acid and include microorganisms (including bacteria, fungi,
yeasts, molds, etc.), viruses, viroids, etc.
[0023] To examine a concentration of bacteria, virus particles, or
other microparticles having nucleic acid, the nucleic acid (DNA,
RNA) contained in the inside must be taken out by efficiently
breaking a cell membrane or a protein envelope and just the nucleic
acid must be fluorescently stained and counted. In the present
invention, electrical discharge is used to rapidly take out the
nucleic acid from the bacteria or virus. In particular, by
performing discharge breakage using the electrode system with the
insulating plate inserted between the mutually opposing electrodes,
the cells can be destroyed within a few minutes to take out the
nucleic acid. This is an extremely short time in comparison to an
enzyme reaction, etc., that breaks the cell membrane.
[0024] An object of the present invention is to perform
concentration measurement of microparticles having nucleic acid in
a short time. By using the discharge breakage, the microparticles
adhered to the microparticle adhesion member can be broken on the
spot. A considerable amount of sample is required to perform a
nucleic acid extraction operation upon transferring the
microparticles having nucleic acid once into solution as is done
conventionally. A microparticle capturing operation that takes a
long time is thus required to collect a large amount of the sample,
and rapid concentration measurement thus could not be
performed.
[0025] On the other hand, by the invention of the present
application, the nucleic acid that diffuses to the surface of the
microparticle adhesion member from the cell or virus that has been
broken at the surface is separated from histones and other proteins
by a surfactant or other charge neutralizer coated on the
microparticle adhesion member. That is, after taking out the
nucleic acid from the microparticles by the discharge breakage, the
nucleic acid is distinguished from other component molecules by
fractionation by electrophoresis. To reduce the time for
electrophoresis, the migration distance must be reduced as much as
possible. In the nucleic acid detection member of the present
invention, the membrane having the thin gel layer on the surface of
the tape is used to electrophorese the nucleic acid in the
thickness direction of the gel layer and thereby fractionate the
nucleic acid from other biopolymers. The nucleic acid that has been
electrophoresed to the tape through the gel layer adheres to the
tape surface. In this state, the nucleic acid has a negative
charge, and thus by using the tape that is made to have the surface
charge of positive polarity, the separated nucleic acid can be
adhered and held reliably on the tape.
[0026] After performing electrophoresis, the gel is peeled off from
the tape. The nucleic acid that is adhered on the tape is then
fluorescently stained. For the fluorescent staining, YOYO, ethidium
bromide, DAPI, or other general dye for fluorescently staining DNA
or RNA may be used. To hold the nucleic acid on the surface
reliably after electrophoresis, a material having a positive
surface charge is preferably used in the tape. Although the number
of the nucleic acid extracted from a single microparticle with
nucleic acid is plural in many cases, the distribution range
thereof is generally within several times a diameter of the
microparticle, and thus by identifying this group by image
processing, counting as the nucleic acid group of one microparticle
can be performed.
[0027] With airborne microparticles, corona discharge is used to
make the microparticles adhere to the surface of the microparticle
adhesion member. The microparticles can thereby be electrically
collected on the membrane surface at high efficiency. Also, where
necessary, the counting precision of the microparticles can be
improved by adjusting the corona discharge voltage or the flow rate
of the air containing the microparticles that are introduced into
the corona discharge field and thereby controlling the adhesion
density of the microparticles having nucleic acid.
[0028] Also, by configuring the microparticle adhesion member and
the nucleic acid detection member as a member in common by
configuring the nucleic acid detection member from the tape and the
gel disposed on one surface side of the tape, the microparticle
adhesion step, the membrane breakage step, the electrophoresis
step, and the nucleic acid measurement step can be performed while
supplying the nucleic acid detection member in a continuous manner,
thereby realizing an extremely rapid measurement system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a process diagram for describing an embodiment in
outline.
[0030] FIG. 2 is a sectional side view of a nucleic acid detection
member for capturing microparticles with nucleic acid.
[0031] FIG. 3 is a schematic view of a corona discharge
apparatus.
[0032] FIG. 4 is a schematic view of a membrane breakage apparatus
for breaking membranes of microparticles.
[0033] FIG. 5 is a schematic view of an electrophoresis apparatus
for making nucleic acid migrate from a gel surface to a tape.
[0034] FIG. 6 is a schematic view of a microparticle adhesion
apparatus for capturing microparticles having nucleic acid by
corona discharge.
[0035] FIG. 7 is a graph of measurement results of number of live
bacteria included among microparticles captured by corona
discharge. In the figure, outline bars indicate results in a case
where a culture medium is set to a positive polarity and solid bars
indicate results in a case where the culture medium is set to a
negative polarity.
[0036] FIG. 8 shows photographs of results of performing a silent
discharge process on Escherichia coli containing the fluorescent
protein GFP in the bodies thereof. It can be understood that by
applying the discharge process, the membranes of the E. coli are
broken so that the GFP contained inside the cells diffuse and the
fluorescent intensity is thereby attenuated largely.
[0037] FIG. 9 is a fluorescence photograph of DNA coated on various
filter surfaces.
[0038] FIG. 10 is a schematic sectional side view of a state where
a microparticle adhesion member and a nucleic acid detection member
are adhered together closely and DNA is electrophoresed.
[0039] FIG. 11 is a photograph of DNA transferred onto a tape of
the nucleic acid detection member after the electrophoresis
process. A dotted line rectangle T indicates a region onto which
reference amounts of DNA were dropped, and a dotted circle S
indicates a region of the electrophoresed DNA. Numerical values
inside T and S indicate DNA amounts.
[0040] FIG. 12 is a fluorescence micrograph image of DNA adhered to
a filter.
[0041] FIG. 13 is an outline diagram of a counting system for
microparticles having nucleic acid.
DETAILED DESCRIPTION OF THE INVENTION
[0042] FIG. 1 is a process diagram for describing an embodiment in
outline. In this process diagram, the microparticle adhesion member
22 and the nucleic acid detection member 23 are configured as
different members. The microparticles 21 having nucleic acid 20
proceed through the steps according to the arrows from an upper
left side of the figure, and by proceeding through the steps at an
upper right side, a lower right side, and a lower left side, the
nucleic acid 20 is measured.
[0043] First, in a microparticle adhesion step at the upper left,
the microparticles 21 having nucleic acid are adhered to the
surface of the microparticle adhesion member 22. The microparticle
adhesion member 22, on which the microparticles 21 are thus
adhered, proceeds to a subsequent membrane breakage step (upper
right of the figure).
[0044] With the microparticles, with which the membranes have been
broken, an electrophoresis step of using a gel 1 and
electrophoresing in a thickness direction of the gel is then
performed to make the nucleic acid 20 migrate from a negative
electrode side toward a positive electrode side. The nucleic acid
20 thus migrates to a surface of a tape 2 of a nucleic acid
detection member 23 (lower right of the figure).
[0045] Finally, the gel 1 on the nucleic acid detection member 23
is removed, the nucleic acid 20 adhered on the surface of the tape
2 is fluorescently stained, and a concentration of the nucleic acid
20 is measured (nucleic acid measuring step).
[0046] Details of the respective steps shall now be described. FIG.
2 is a sectional side view of the nucleic acid detection member 23
for electrophoresing the microparticles 21. The nucleic acid
detection member 23 has, as a substrate, the plastic tape 2 formed
from a non-woven fabric that is surface treated with an amino group
(NH.sub.2.sup.+) to have a surface charge of positive polarity, and
has on the surface thereof a membrane formed by coating the agarose
gel 1 to a thickness of not less than approximately 1 mm. The gel 1
is used to electrophorese the microparticles 21 having nucleic
acid. The nucleic acid detection member 23 that is configured as a
tape-like membrane is stored in a cartridge and can be supplied in
a continuous manner. For example, the tape may be approximately 1
mm to 5 mm in width. The nucleic acid detection member 23 can be
used in common as the microparticle adhesion member 22. That is,
after adhering the microparticles 21 on the surface of the agarose
gel 1, the membranes of the microparticles 21 are broken, and then
electrophoresis is performed to make the nucleic acid 20 migrate in
the direction of the tape 2 and adhere to the surface of the
tape.
[0047] A corona discharge apparatus (microparticle adhesion
apparatus) 24, shown in FIG. 3, can be used to make microparticles
having nucleic acid that are suspended in air adhere to a tape-like
membrane. The corona discharge apparatus 24 includes a needle-like
high-voltage electrode 3 and a flat plate ground electrode 9. A
distance between the electrodes 3 and 9 is set to an interval of
approximately 5 mm to 10 mm, and the microparticle adhesion member
22 is supplied to a surface of the flat plate ground electrode
9.
[0048] To use the tape-like nucleic acid detection member 23 to
adhere microparticles having nucleic acid that are adhered to a
wall or clothing, etc., the nucleic acid detection member 23 is
drawn out from the cartridge and the surface of the gel 1 is
directly contacted with the wall or clothing, etc. Or, the
membrane-like microparticle adhesion member 22 (or the nucleic acid
detection member 23 serving in common as the microparticle adhesion
member 22) of a diameter of approximately several mm may be
prepared separately and put in contact with the wall or clothing,
etc., and the subsequent processes of nucleic acid extraction,
fluorescence staining, etc., may be performed thereafter.
[0049] FIG. 4 shows a membrane breakage apparatus 25 that can be
used in the membrane breakage step. The membrane breakage apparatus
25 includes mutually opposing silent discharge electrodes 5 and 19,
an insulating plate 6 inserted between the two electrodes, and a
member mounting space 26 into which the microparticle adhesion
member 22 is inserted. The two discharge electrodes 5 and 19 are
configured as parallel plate electrodes, and by disposing the
insulating plate 6 at the electrode at one side, a gap of the
member mounting space 26 is set to not more than approximately 2
mm. The electrode 5 at the upper side is configured from a
stainless-steel mesh, and the electrode 19 at the lower side is the
ground electrode. A high-voltage alternating power supply 4 is
connected to the electrode 5. Also, a spacer 7 of predetermined
thickness is sandwiched between the insulating plate 6 and the
electrode 9. An alternating voltage, for example, of 30 kHz and
approximately 10 kV is applied to generate a silent discharge.
[0050] After the microparticles 21 having the nucleic acid 20 are
adhered to the microparticle adhesion member 22, the microparticle
adhesion member 22 is inserted into the member mounting space 26,
and the membranes of the microparticles 21 are broken by the silent
discharge.
[0051] Subsequently, the nucleic acid 20 is extracted by adding SDS
or other surfactant to the surfaces of the membranes of the broken
microparticles 21 having the nucleic acid 20 that are adhered to
the surface of the microparticle adhesion member 22. Thereafter, an
electrophoresis apparatus 27, shown in FIG. 5, is used to perform
electrophoresis of the nucleic acid 20. The electrophoresis
apparatus 27 includes a container 30, in which is placed an aqueous
solution (electrolyte solution 10) that has conductivity, and a
pair of positive and negative electrodes 28 and 29 disposed at a
bottom surface and an upper surface of the container 30. The
nucleic acid detection member 23 is inserted between the two
electrodes 28 and 29 to perform electrophoresis of the nucleic acid
20. The electrophoresis is performed in the direction of thickness
of the gel 1 with the gel 1 side of the nucleic acid detection
member 23 being negative and the tape 2 side being positive, and
the nucleic acid 20 is electrophoresed from the gel 1 surface to
the tape 2. The time required for electrophoresis is normally about
several minutes.
[0052] After performing the electrophoresis, the nucleic acid
detection member 23 is removed from the electrophoresis apparatus
27, and the gel 1 is peeled off by a scraper to leave just the tape
2 on which the nucleic acid 20 is adhered. Thereafter, the tape 2
is passed through a container in which a fluorescent dye solution
is placed to fluorescently stain the adhered nucleic acid 20.
[0053] Thereafter, excitation light is irradiated on the tape 2 to
make fluorescence be emitted, and the nucleic acid 20 that emits
fluorescence is counted.
Examples
[0054] Capture of Microparticles having Nucleic Acid by Corona
Discharge
[0055] A microparticle adhesion apparatus including a corona
discharge electrode, shown in FIG. 6, was used to capture bacteria
suspended in air. The apparatus includes a needle-like,
high-voltage electrode 3' for corona discharge, a culture medium 13
serving as a flat plate ground electrode (collecting electrode),
and a high-voltage DC power supply 15. The symbol 13A indicates an
electrode for grounding the culture medium 13. A fan 12 is disposed
at an upper surface side of the apparatus. By driving the fan 12,
air in a room is introduced from the upper surface side (symbol 11)
and discharged from a lower surface side (symbol 14). When the air
passes between the two electrodes 3' and 13, the microparticles
with nucleic acid are captured by the culture medium 13.
[0056] FIG. 7 shows counting results of number of colonies
resulting from capturing microparticles using the apparatus. Indoor
air was fed at a rate of 18 liters per second to the lower side by
the fan 12, and corona discharge was generated across the silver
needle electrode 3' and the culture medium 13. The numbers of
colonies observed were counted upon subjecting the culture medium
13 to culturing for 2 days after performing corona discharge for 10
minutes with the culture medium 13 side being set to positive or
negative polarity and the voltage being varied from 0 kV to 30
kV.
[0057] As a result, it was confirmed that (1) in both cases of
using positive polarity and negative polarity, the number of
bacteria captured is increased by not less than 10 times by the
corona discharge for ten minutes (22 times in the case of negative
polarity and 47 times in the case of positive polarity), and that
(2) when corona discharge of positive polarity is used, a capture
efficiency of live bacteria is improved in comparison to the case
where corona discharge of negative polarity is used. It is
considered that the number of bacteria colonies was greater when
corona discharge from the positive electrode was performed because
generation of ozone, which is harmful to bacteria, is suppressed in
the case of atmospheric corona discharge at the silver positive
electrode.
Breakage of Bacteria by Silent Discharge
[0058] FIG. 8 shows photographs of Escherichia coli after breakage
of membranes. For fluorescence to be generated, the E. coli used
has introduced therein a gene that produces the jellyfish-derived
fluorescent protein GFP. The microparticle adhesion member 22,
having the E. coli coated thereon, was set in the membrane breakage
apparatus 25 including the silent discharge electrodes 5 and 19
shown in FIG. 4, and an alternating voltage of 30 kHz and 25 kVpp
was applied. When the cell membranes of E. coli are broken, the GFP
contained in the cells diffuses, and the breakage can thus be
confirmed by a large attenuation of the fluorescence intensity.
Even from a fluorescence photograph of a sample in which E. coli
not subject to the silent discharge process and E. coli broken by
the silent discharge process are mixed, was confirmed that the
fluorescence intensity is attenuated largely by the silent
discharge process. Although an enzyme reaction is normally used for
breaking the cell membranes of E. coli, this process requires
several hours, and thus the breakage of bacteria by silent
discharge has a characteristic that it can be performed in an
extremely short time.
Electrophoresis of DNA
[0059] FIG. 9 is a fluorescence photograph of a case where a DNA
molecule group is coated on the surface of the microparticle
adhesion member 22. As shown schematically in FIG. 10,
electrophoresis of DNA was performed by the electrophoresis
apparatus 27 upon putting the nucleic acid detection member 23 in
close contact with the surface side of the microparticle adhesion
member 22 on which the DNA molecules were coated (lower surface
side in the figure).
[0060] FIG. 11 shows results of fluorescently staining the tape 2
of the nucleic acid detection member 23 after the electrophoresis.
By measurement of the DNA amount by staining, it was confirmed that
approximately 30 to 50% of the DNA molecules migrated from the
microparticle adhesion member 22 and became adhered to the tape 2
by the electrophoresis. FIG. 12 is a fluorescence micrograph image
of the DNA adhered to the tape 2. It was thus demonstrated that
individual DNA molecules can be identified. The electrophoresis
time is only a few minutes, and it was demonstrated that DNA can be
sampled through the thin gel.
System for Counting Microparticles with Nucleic Acid
[0061] FIG. 13 shows, in outline, a counting system according to a
present embodiment. This system has a configuration where the
nucleic acid detection member 23 is used in common as the
microparticle adhesion member. The nucleic acid detection member 23
is manufactured in a continuous, band-like manner and is subject to
the microparticle adhesion step (A), the membrane breakage step
(B), the electrophoresis step (C), and the nucleic acid measurement
step (D) by moving at a predetermined speed from a left side to a
right side of the figure.
[0062] That is, the nucleic acid detection member 23 passes through
the corona discharge apparatus 24 with the gel 1 being faced
upward. The microparticles 21 with the nucleic acid become adhered
to the surface of the gel 1 in this process. When the nucleic acid
detection member 23 then passes through the inside of the member
mounting space 26 of the membrane breakage apparatus 25, the
membranes of the microparticles 21 are broken by silent
discharge.
[0063] When the nucleic acid detection member 23 then passes
through the electrophoresis apparatus 27, the nucleic acid 20
migrates through the inside of the gel 1 as it is electrophoresed
from the negative electrode side toward the positive electrode side
and becomes adhered to the surface of the tape 2.
[0064] After passage through the electrophoresis apparatus 27, the
gel 1 of the nucleic acid detection member 23 is peeled off,
leaving just the tape 2, and thereafter, the nucleic acid 20 is
measured by irradiation from the nucleic acid detection apparatus
28 that generates the excitation light.
[0065] Thus, by the present invention, it has become possible to
perform from the capture of the microparticles 21 having the
nucleic acid 20 to the breakage of the microparticles 21,
extraction of the nucleic acid 20, the fixing of the nucleic acid
to the tape 2 surface by electrophoresis, fluorescent staining, and
counting within 10 minutes.
INDUSTRIAL APPLICABILITY
[0066] By the present invention, the evaluation of circumstances of
contamination by microorganisms and viruses of air, wall surfaces,
and floors of hospitals and food factories or utensils and clothing
used for surgery and food manufacturing can be performed within 10
minutes, thereby enabling ascertainment of danger of infection or
taking of measures necessary for preventing infection to be
performed substantially at real time. and application to sanitation
control is thus anticipated.
DESCRIPTION OF REFERENCE NUMERALS
[0067] 1 agarose gel [0068] 2 plastic membrane with positive charge
[0069] 3, 3' needle electrode [0070] 5 discharge electrode [0071] 6
insulating plate [0072] 9, 19 ground electrode [0073] 13 culture
medium (flat plate ground electrode) [0074] 20 nucleic acid [0075]
21 microparticle [0076] 22 microparticle adhesion member [0077] 23
nucleic acid detection member [0078] 24 microparticle adhesion
apparatus [0079] 25 membrane breakage apparatus [0080] 26 member
mounting space [0081] 27 electrophoresis apparatus [0082] 28
nucleic acid detection apparatus
* * * * *